Abstract

Phorbol esters induce apoptosis in androgen-sensitive LNCaP cells, which
express neutral endopeptidase (NEP), but not in androgen-independent
prostate cancer (PC) cells, which lack NEP expression. We investigated
the role of NEP in PC cell susceptibility to
12-O-tetradecanoylphorbol-13-acetate (TPA). Western
analysis showed that expression of NEP and protein kinase Cδ (PKCδ)
correlated with PC cell sensitivity to TPA-induced growth arrest and
apoptosis in LNCaP cells and in TSU-Pr1 cells expressing an inducible
wild-type NEP protein. Inhibition of NEP enzyme activity using the
specific NEP inhibitor CGS24592, or inhibition of PKCδ using
Rottlerin at concentrations that inhibit PKCδ but not PKCα,
significantly inhibited TPA-induced growth inhibition and cell death.
Furthermore, pulse-chase experiments showed PKCδ is stabilized in
LNCaP cells and in TSU-Pr1 cells overexpressing wild-type NEP compared
with PC cells lacking NEP expression. This results from NEP
inactivation of its neuropeptide substrates (bombesin and
endothelin-1), which in the absence of NEP stimulate cSrc kinase
activity and induce rapid degradation of PKCδ protein. These results
indicate that expression of enzymatically active NEP by PC cells is
necessary for TPA-induced apoptosis, and that NEP inhibits
neuropeptide-induced, cSrc-mediated PKCδ degradation.

Introduction

TPA
3
exerts numerous effects on cells, including
proliferation, malignant transformation, differentiation, and cell
death
(1, 2)
. These effects are mediated in part through
modulation of PKC isoenzymes. Numerous investigators have shown that
phorbol esters can induce apoptotic cell death in androgen-sensitive PC
LNCaP cells but not in androgen-independent PC-3 or DU145 cells
(3,
4,
5)
. Powell et al.(6)
reported
that TPA-induced cell death in LNCaP cells correlated with increased
expression of PKCα mRNA, but not other PKC isoforms, and with
translocation of PKCα to non-nuclear membranes. However, Fujii
et al.(7)
recently reported that PKCδ
mediates phorbol ester-mediated apoptosis in LNCaP cells, demonstrating
that phorbol ester-induced cell death can be partially (∼50%)
blocked by a PKCδ-inhibitor or a dominant-negative PKCδ mutant. The
biological effect of PKCδ is cell-type specific, and overexpression
can both inhibit cell growth
(8)
or enhance
anchorage-independent growth and metastatic potential
(9)
.
PKCδ activity appears to be regulated in part through tyrosine
phosphorylation by Src kinase, which results in degradation of PKCδ
protein
(10)
.

LNCaP cells express neutral endopeptidase 24.11 (NEP, CD10, CALLA, EC
3.4.24.11), a Mr
90,000–110,000 zinc-dependent cell-surface metallopeptidase,
whereas androgen-independent PC cell lines do not
(11)
.
NEP can regulate through its enzymatic function access of neuropeptides
such as bombesin, neurotensin, and ET-1 to their cell-surface
G-protein-coupled receptors. Neuropeptide signaling involves activation
of cSrc kinase activity, which in turn leads to phosphorylation of
several downstream substrates such as focal adhesion kinase and p130Cas
(12,
13,
14)
. Mari et al.(15)
reported previously that a catalytically active NEP protein is required
for phorbol ester-induced growth arrest in Jurkatt T cells. In the
present study, we considered whether NEP and its substrates were
involved in regulating the expression of PKCδ in LNCaP cells and
whether NEP and PKCδ expression were required for TPA-induced
apoptosis. We report that ET-1 and bombesin induce PKCδ
down-regulation caused by rapid PKCδ degradation in PC cells, which
is mediated by cSrc kinase activation, and that PKCδ down-regulation
is blocked by NEP in LNCaP cells, which facilitates TPA-induced
apoptosis.

Cell Growth Assays.

PC cells (1 × 10
4
/well) were
plated in 12-well tissue culture plates (Falcon Division, Becton
Dickinson, Cockeysville, MD). After overnight culture in regular media
(LNCaP, TSU-Pr1, DU145, and PC-3) or culture for 48 h in media
with or without tetracycline (WT-5, TN-12, and M-22), cells were
treated with various reagents for 48 h. Cells were harvested and
counted using a Coulter Counter ZM (Coulter Electronics, Hialeah, FL).
Each data point represents the average cell number of triplicate
samples from a single experiment. Statistical analyses were performed
using an unpaired t test. Ps less than 0.005 are
reported as <0.005. All growth assays were performed on three separate
occasions with similar results.

Apoptosis Assays.

Early apoptotic cells were detected using the Annexin V apoptosis
detection kit (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Briefly, cells evenly distributed in Lab-Tek chamber slides (Nalge Nunc
International, Naperville, IL) were treated with various
reagents for 24 h. Cells were washed twice with cold PBS, washed
once with 1 × Assay Buffer and with 1 μg of Annexin
V-FITC with 500 μl of 1 × Assay Buffer added.
Propidium iodide (0.5 μg ) was added to each well for nuclear
counterstain. After incubation for 15 min at room temperature in the
dark, positively stained cells were enumerated using a fluorescence
microscope at ×100–400. Each data point represented the average cell
number in six independent microscopic fields of a single experiment.
The statistical analysis was performed using an unpaired t
test. Ps <0.05 were regarded as statistically significant.
All assays were performed on three separate occasions with similar
results.

For cell cycle analysis, cells were fixed in 70% ethanol and stained
with 50 μg/ml propidium iodide. Cell cycle progression and apoptosis
were analyzed by flow cytometry using a Becton Dickinson
fluorescence-activated cell sorting system. Twenty thousand
events were recorded for each treatment.

Pulse-Chase Assay.

An equal number of cells were cultured in RPMI 1640 lacking methionine
for 30 min, in the same media containing 300 μCi/ml[
35S]methionine for 1 h, and washed with
PBS and then in RPMI supplemented with 10% FCS and 0.15 mg/ml
nonradioactive methionine for specific time periods. Cells were lysed
in RIPA buffer. For immunoprecipitation, 300-μg lysates were
incubated for 1 h with 1 μg of anti-PKCδ antibody and then for
1 h with 40 μl of protein G-Sepharose beads (Amersham Pharmacia)
at 4°C. Immunoprecipitates were collected by centrifugation at
12,000 × g for 1 min, washed with RIPA
buffer, and resuspended in 2 × Laemmli sample buffer.
Samples were resolved on an 8% SDS-PAGE and transferred to
nitrocellulose. Autoradiography and immunoblotting were performed using
the same membrane. The relative intensity of each band obtained by
autoradiography was measured by NIH image.

Results

Western blot analysis revealed high levels of NEP and PKCδ proteins
in total cell lysates derived from LNCaP cells but not in lysates
derived from androgen-independent TSU-Pr1, DU145, or PC-3 cells (Fig. 1A⇓
, panel 1 and panel 2). In comparison,
PKCα protein was expressed at lower levels in LNCaP cells relative to
other PC cell lines (Fig. 1A⇓
, panel 3), as
reported previously
(6)
. In contrast to parental
TPA-sensitive LNCaP cells, NEP and PKCδ proteins could not be
detected in a TPA-resistant subclone of LNCaP, LN10H cells
(6)
, whereas PKCα protein was expressed at similar
levels.

Increased PKCδ expression and TPA-induced growth
inhibition in PC cells expressing functional NEP. A,
total cell lysates (20 μg) from PC cells were analyzed for NEP
protein by Western blot as described in “Materials and Methods”
using the anti-NEP mAb NCL-CD10–270 (panel 1),
anti-PKCδ Ab (panel 2), anti-PKCα Ab
(panel 3), and anti-actin Ab (panel
4). B, cell growth assays were performed with
the addition of the complex indicated (10 nm TPA, 10μ
m Rottlerin, and 100 nm Gö6976) as
described in “Materials and Methods.” Bars, SD.
C, LNCaP cells were treated with or without 100
nm CGS24592 for 16 h, and the expressions of PKCδ
(panel 1) and actin (panel 2) were
determined by Western blot. D, NEP-inducible TSU-Pr1
clones were treated with or without tetracycline (Tet.)
for 48 h and analyzed for NEP protein by Western blot using
anti-NEP mAb (panel 1), anti-PKCδ Ab (panel
2), anti-PKCα Ab (panel 3), and anti-actin Ab
(panel 4). E, cell growth assays were
performed in media containing FCS with the addition of 10
nm TPA with or without 50 μm Rottlerin.
Bars, SD.

Incubation of PC cells in media containing FCS plus 10 nm
TPA for 48 h induced ∼80% growth inhibition in LNCaP cells
compared with untreated control (P < 0.005)
but not in TSU-Pr1, DU145, or PC-3 cells (Fig. 1B)
⇓
. Both
PKCα and PKCδ have been implicated in TPA-induced growth inhibition
of LNCaP cells
(6, 7)
. We therefore assessed the effects
on TPA-induced growth inhibition of the PKC-inhibitors Rottlerin
(IC50 = 3–6 μm
for PKCδ, IC50 = 40μ
m for PKCα, PKCβI, and PKCγ) at a
concentration which selectively inhibits PKCδ, and Gö6976
(IC50 = 2–6 nm for PKCα and
PKCβI; no inhibition at μm concentrations for
PKCδ) as a PKCα inhibitor. As illustrated in Fig. 1B⇓
,
TPA-induced growth inhibition of LNCaP cells was reversed by
pretreatment with 10 μm Rottlerin 2 h
before TPA (P < 0.005) but not by 100
nm Gö6976. No significant effect of either
inhibitor was observed in TSU-Pr1, DU145, and PC-3 cells. Taken
together, these results show that TPA sensitivity correlates with NEP
and PKCδ expression in PC cells and support previous studies which
suggest that TPA-induced growth inhibition in LNCaP cells is mediated
by PKCδ.

To assess whether NEP is needed for PKCδ expression and TPA
sensitivity in PC cells, we cultured LNCaP cells in media containing
FCS with the addition of the specific NEP enzyme inhibitor CGS24592 at
a concentration of 100 nm, which completely inhibits NEP
enzyme activity
(14)
, and found that PKCδ protein levels
were significantly less than control-treated LNCaP cells (Fig. 1C)
⇓
. Next we examined PKCδ protein expression in TSU-Pr1
cells containing a tetracycline-repressed (tet-off) inducible wild-type
NEP (WT-5 cells), catalytically inactive NEP (M-22 cells), which
contain a point mutation in the zinc-binding domain required for NEP
enzymatic function
(14)
, and control (empty vector; TN-12
cells). Western blot analysis (Fig. 1D⇓
, panel 1)
and enzyme studies confirmed NEP protein expression in both WT-5 and
M-22 cells, but not in control TN-12 cells, 48 h after withdrawal
of tetracycline from the media, whereas high levels of NEP-specific
activity could be detected only in total cell lysates from WT-5
cells (not shown; see Ref.
14
). High levels of
PKCδ protein expression were present in cells expressing wild-type
NEP proteins (WT-5), whereas barely detectable PKCδ protein was
detected in cells which did not express NEP (tet-repressed WT5, TN-12)
or which expressed catalytically inactive NEP proteins (M-22; Fig. 1D⇓
, panel 2). In contrast, PKCα expression was
not affected by NEP expression or tetracycline in these cell lines
(Fig. 1D⇓
, panel 3). Cell growth assays showed
that culturing in media containing FCS with 10 nm
TPA for 48 h after expression of wild-type cell-surface NEP
resulted in a >60% decrease in cell number in WT-5 cells
(P < 0.005) but did not alter cell growth in
TN-12 cells or in M-22 cells expressing catalytically inactive NEP
(Fig. 1E)
⇓
. Similar to experiments using LNCaP cells,
pretreatment with 10 μm Rottlerin 2 h
before 10 nm TPA treatment in NEP-expressing WT-5
cells reversed TPA-induced growth inhibition in TPA-treated WT-5 cells
(P < 0.005; Fig. 1E⇓
). Rottlerin
alone had no significant effect on these cells (data not shown). Taken
together, these results suggest that the expression of catalytically
active NEP protein is required for increase in PKCδ protein
expression in PC cells, and that PKCδ mediates susceptibility of PC
cells to TPA-induced growth inhibition.

Discussion

The results presented here help clarify previous reports on
phorbol ester-induced cell death in androgen-sensitive LNCaP cells but
not in androgen-independent PC cells. Our data suggest that
neuropeptides such as ET-1 and bombesin stimulate cSrc kinase activity,
which in turn phosphorylates PKCδ and leads to rapid degradation of
PKCδ protein. TPA-induced apoptosis is mediated through
PKCδ. Thus, androgen-independent PC cells, which do not express NEP,
are resistant to TPA treatment because they express low levels of
PKCδ protein. In contrast, LNCaP cells constitutively express NEP.
NEP inactivates through hydrolysis neuropeptides such as ET-1 and
bombesin, leading to diminished cSrc kinase activity and stable
expression of PKCδ protein. Consequently, PKCδ-expressing LNCaP
cells are extremely sensitive to TPA-induced apoptosis. Although a
previous study reported that NEP is required for TPA-induced growth
arrest in Jurkatt T cells
(15)
, the mechanism of NEP in
allowing susceptibility to TPA had not been elucidated. Our results
highlight the involvement of NEP in TPA-induced apoptosis mediated by
PKCδ in PC cells.

Our studies indicate that TPA-induced apoptosis and growth
inhibition in LNCaP cells is predominantly mediated by PKCδ rather
than by PKCα. Henttu et al.(5)
have reported
that calcium-independent PKC isoenzymes such as PKCδ, rather than
PKCα, are predominantly activated in TPA-treated LNCaP cells, which
supports our studies. Recent reports also implicate PKCδ as a
proapoptotic kinase
(19,
20,
21)
. A proteolytic cleavage site
for caspase-3 has been identified at the V3 (hinge) region of PKCδ
with cleavage resulting in the release of an active
Mr 40,000 fragment corresponding to
the PKCδ COOH-terminal kinase domain
(19)
. However,
similar to a previous report
(7)
, pretreatment with the
selective caspase-3 inhibitor DEVD-CHO showed little inhibitory
effect on TPA-induced cell death in LNCaP or WT-5 cells (data not
shown). These results suggest that the TPA-induced apoptotic pathway
mediated by PKCδ in PC cells is independent of caspase-3 activity or
caspase-3-mediated PKCδ cleavage, and that PKCδ can act as a
primary effector or is involved in other pathways for apoptosis via its
allosteric activation
(7)
. Recent studies show that
translocation of PKCδ holoenzyme, and not its catalytic fragment,
onto mitochondria induces cytochrome c release and
apoptosis, and that this translocation precedes the activation of
caspases
(22, 23)
. This suggests that proteolytic cleavage
may not be required for PKCδ kinase activation and apoptosis
induction. Others have suggested that various cell cycle regulators
(3)
or ceramide
(4)
mediate TPA-induced
apoptosis in LNCaP cells. We have found NEP expression in WT-5 cells
up-regulates p21WAF/CIP1 expression and inactivates the retinoblastoma
protein, which induces
G0-G1
arrest,
4
leading us to speculate on the possibility that PKCδ stabilized by
NEP can affect these cell cycle regulators via its allosteric
activation.

NEP neuropeptide substrates such as ET-1 and bombesin can act as
survival and antiapoptotic factors
(24, 25)
,
transactivators of epidermal growth factor receptor
(26)
,
and activators of Akt/protein kinase B cell survival pathway
(27, 28)
. As a regulator of these peptides to their cell surface
receptors, NEP is involved in various critical signaling pathways. Our
data for the first time define one mechanism of NEP function as an
inducer of apoptosis through stabilization of PKCδ expression. PKCδ
activity has been implicated in mediating apoptosis in response to
various antitumor reagents such as etoposide
(20)
and
cis-platinum
(29)
as well as TPA. Thus, through
its ability to inhibit various cell survival pathways by inactivating
mitogenic neuropeptides, NEP may be a potential therapeutic modality to
use in combination with various agents to treat prostate cancer.

Acknowledgments

We thank Dr. Thomas Powell for useful discussions and for
supplying the LN10H PC cell line, and Catherine Kearney and Lana
Winter for secretarial assistance.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵1 This work was supported by NIH Grant CA 80240,
the Association for the Cure of Cancer of the Prostate (CaP CURE), and
the Dorothy Rodbell Foundation for Sarcoma Research. J. D. is a
recipient of a Department of Defense Prostate Cancer Research Program
Post-doctoral Traineeship Award.